of of of of of

DIETARY FAT INTAKE DURING PRE- AND POST-WEANING TIME PERIOD AND ITS ASSOCIATION WITH THE ONSET OF CARDIOVASCULAR

DISEASE IN THE OFFSPRING

By

Kanta Chechi, M.Sc

A thesis submitted to the School of Graduate studies

in partial fulfillment of

the requirements for the degree of

Doctor of Philosophy

Department of Biochemistry, Faculty of Science

Memorial University June 2010

St. John's Newfoundland & Labrador

ABSTRACT

The 'fetal origins' of cardiovascular disease (CVD) hypothesis proposes that maternal nutritional environment during pregnancy can play an important role in determining the cardiovascular health of an individual in adult life. A typical Western diet is rich in dietary fats, a fact that has been linked to the increased prevalence of CVD. In addition to the quantity of fat, the quality of fat is also known to affect the development of CYD.

Whilst an increased consumption of saturated fatty acids (SFA) has been associated with higher incidence of CYD, a diet rich in polyunsaturated fatty acid (PUF A) has been suggested to lower the risk of developing CYD. Considering that nutrition patterns are shifting towards a higher-fat Western diet globally, it is of interest to understand the role of a high-fat maternal diet in the fetal origins of CYD. The current thesis was designed to understand the role of the quantity, a well as the quality, of maternal dietary fat intake during pregnancy, in the fetal origins of CYD in the adult offspring. In addition, the role of interaction between the pre- and post-natal dietary fat intake on the offspring health was assessed. Early programming experiments were conducted using C5781/6 mice, which have been extensively used as an animal model to investigate the dietary fat- mediated regulation of lipid metabolism. Lipid metabolism and aortic vascular function were chosen as the study outcomes to estimate the risk of developing CYD in the offspring. Results indicated that a high-fat maternal diet rich in SFA (lard) was associated with a reduced expression of hepatic low-density lipoprotein (LDL)-receptor and a higher concentration of LDL-cholesterol in the offspring. On the other hand, a high-fat maternal diet rich n-6 PUFA (safflower oil) was associated with higher mRNA expression of hepatic lecithin: cholesterol acyltransferase and higher concentration of high-density

lipoprotein-cholesterol in the offspring. The high-fat maternal diet, irrespective of the type of fat, however was associated with reduced aortic contractile reactivity towards KCI, phenylephrine and thromboxane mimetic U44619, in the female offspring. In addition, significant interaction of pre- and post-weaning diets was reported for various outcomes studied in the offspring, suggesting the importance of both prenatal and postnatal environments in regulating the offspring cardiovascular health.

In a separate study, the effects of n-3 PUF A-rich flax oil feeding were assessed on various parameters associated with metabolic syndrome, using the SHRJNDmcr-cp rat model. Flax oil feeding was associated with significantly lower hepatic triglycerides and cholesterol concentrations in the obese rats. In addition, flax oil feeding was associated with lower plasma insulin concentrations and oxidative stress in the obese rats. An up regulation in the hepatic expression of peroxisome-proliferator activated receptor-y (PPAR-y) was found to be negatively correlated with the hepatic TG and cholesterol concentrations in the obese rats, thus pointing towards the activation of PPAR-y dependent pathways behind the hepatic lipid-lowering effects offlax oil supplementation.

Taken together, the results presented in the current thesis support the role for the quantity and the quality, of dietary fats consumed during pre- and post-weaning time periods, on the development of key parameters associated with the onset of CVO.

CO-AUTHORSHIP STATEMENT

For the work presented in chapter-3, published in American Journal of Physiology, Regulatory, Integrative and Comparative Physiology (2009) 296: R1029-40, I, Kanta Chechi, was involved with the design of the study, conducting the experiments, analyzing the data and preparation ofthe manuscript.

For the work presented in Chapter-4, which is under review for publication in the Briti h Journal of Nutrition, I was involved with the design of the study, conducting the experiments, analyzing the data and preparation of the manuscript.

For the work presented in Chapter-S, published in Experimental and Clinical Cardiology (2006) 11:129-135, I was involved with the design of the study, conducting the experiments, analyzing the data and preparation ofthe manuscript.

For the work presented in Chapter-6, which is accepted for publication in Prostaglandins, Leukotrienes and Essential Fatty acids 2010 (in press), I was involved with the design of the study, conducting the experiments, analyzing the data and preparation of the manuscript.

For the work presented in Chapter-7, published in the British Journal of Nutrition (2010) (Epub ahead ofprint, doi: 10.1017/S0007114510002187), I was involved with the design of the study, conducting the experiments, analyzing the data and preparation of the manuscript.

I wisfi to eJ(press my sincere appreciation to my supervisor ([)r. Suk_fiinaer 'l(_aur Cfieema ana supervisory committee mem6ers ^(j)r. CR.p6ert (}3ertoCo ana ^(j)r. qene Jferz6erg for tlieir continuous guiaance ana support auring tfie course of my researcfi work_ ana tfie preparation

of

of

(}3iomedica[ Sciences witfi tfie vascufar studies. )I friena[y environment in fiis [a6 was very fie[pfu~

especia[[y auring Cong fiours of myograpfi-eJ(periments.

'11iankj to tfie mem6ers of tfie fa6, (}3iocfiemistry ([)epartment ancf a[[ my frienas incfuding Prati6fia, )I[Rg. di, )Inura, Ju[ia, Jfifary, rM_ary, !Nancy, CR.p6in, ¹Erica, ~nol<fz, Cfiaritfia, Sampatfi, rJ?.slniru, )I[ison, (/)anieffe, (}3arry, Simone, (/)anna, CR.p6ert, Semone, 'l(austa6fi, }lfe.:K, Sarafi, )Irpan, Satty, Iti, 1/ai6fiav, Satomi, !Naomi, rtuRg.ri, to name a few, some of wfiicfi surrounaea me aurina my qracfuate studies fiere at rM_emoria~ some in Japan ana in India. '11ieir association, Cove, encouragement ana support not just as co[feagues, 6ut afso as my cCose frienas is sometfiing tfiat I wi[[ cfierisfi for tfie rest of my fife.

'Witfi aeep sense of gratituae ana reverence, I wouU Ei~ to tfiank_ (j)r. Jofin (}3rosnan, ([)r. rM_argaret (}3rosnan, (/)r. CR.pss rM_cqowan, ([)r. rtuk_io rtamori, (j)r. 'l(_atsumi I~aa, (j)r. 'l(risfina 'l(umar )Iggarwa[

ana (/)r. Satomi 'J(Jlgota as my mentors, not on[y in tfie researcfi environment, 6ut afso as tfie peopfe wfio taugfit me tlie va[ues of fiara war~ encouragement, warmtli ana Rj.naness in fife.

I wouU Ei~ to tliank_Sclioo[ of qraauate studies, rM_emoria[ Vniversity for proviaing tlie financia[

support tlirougliout tlie program. I wouU afso Ei~ to tfiank_ tlie rM_atsumae Intemationa[ Pounaation for providing me a fdCowsfiip to conauct a sfiort researcfi project in Japan.

I wisfi to aedicate tliis work_ to my parents, fami{y ana my fius6ancf, for witliout tfieir unconditiona[

[ave, support ana constant encouragement; tliis journey wouU not fiave 6een possi6fe.

TABLE OF CONTENTS

ABSTRACT ... ii

CO-AUTHORSHIP STATEMENT ... iv

;zLCJ\.JI{OWLr:ECD(i?rffEJ.fl'S ... v

TABLE OF CONTENTS ... vi

LIST OF TABLES ... xiii

LIST OF FIGURES ···XV LIST OF ABBREVIATIONS ... xvi

LIST OF APPENDICES ... xx

TABLE OF CONTENTS CHAPTER 0 NE ... 1

Introdztction and Overview ... 1

1.1 CARDIOVASCULAR DISEASE ............... 2

1.2 FETAL ORIGINS HYPOTHESIS ...................... 6

1.2.1 Thrifty phenotype hypothesis ... 8

1.2.2 Predictive adaptive response hypothesis ... 9

1.3 MATERNAL UNDER-NUTRITION AND FETAL ORIGINS OF CVD: ANIMAL STUDIES ................................... I 0 1.3.1 Fetal tissue remodeling .................................. II 1.3.2 Fetal glucocorticoid exposure ............................. 12

1.3.3 Epigenetic mechanisms ................................ 13

1.4 DIETARY FATS AND FETAL ORIGINS HYPOTHESIS ................ 15

1.4.1 Dietary fat consumption and fetal origins of CVD: Animal Studies ... 16

1.4.1.1 Dietary fats andfetal origins ofdyslipidemia .................. 16

1.4.1. 2 Dietary fats and fetal origins of endothelial dysfunction .... 20

1.4.1.3 Dietary fats andfetal origins ofhypertension ............ 23

1.4.1.4 Dietary fats and fetal origins of obesity .................... 26

1.4.1.5 Dietary fats and fetal origins of insulin resistance and Type 2DM ...... 31

1.5 DIETARY FAT-MEDIATED REGULATION OF LIPID METABOLISM ... 33

1.5.1 Dietary fats and lipoprotein metabolism ........................... 33

1.5.2 Abnormal lipid metabolism and onset ofCVD ......................... 34

1.5.3 Dietary fat-mediated regulation of gene expression ....... 38

1.5.3.1 Dietary fats and PPAR 's ............................... 38

1. 5. 3. 2 Dietary fats and SREBP 's ................................ 40

1.6 RATIONALE ....................... 42

1.7 AIMS AND OBJECTIVES ......................... 42

CHAPTER TWO ... 46

General Methodology and Breeding Outcomes .... 46

2.1 ANIMALS AND DIETS ..................................... 47 2.1.1 Diets .......................................... 47

2.1.2 Breeding and maintenance of animals .................. 47

2.1.2.1 Pregnancy rate, pup survival rate and sex ratio of the pups at the time of weaning .................................. 51

2.1.2.1.1 Pregnancy and pup survival rate ....................... 51

2.1.2.1.2 Sex ratio of the pups ..................................... 54

2.2 PLASMA LIPID ANALYSIS .................................... 56

2.3 PLASMA FF A AND BLOOD GLUCOSE ANALYSIS .............. 56

2.4 VASCULAR FUNCTION ANALYSIS ...................... 57

2.5 GLC ANALYSIS ...................................... 57

2.6 STATISTICAL ANALYSIS ............................ 58

CHAPTER THREE ... 59

Effects of feeding a high-fat maternal diet rich in lard (SFA) during gestation and lactation on lipid metabolism and aortic vascular function in the adult offspring ... 59

3.1 INTRODUCTION .................................................... 60

3.2 METHODS ............................................. 62

3.2.1 Experimental design .......................................... 62

3.2.3 Vascular function analysis .................... 64

3.2.4 Calculations and Statistical Analysis ..................... 64

3.3 RESULTS ................................................................. 65

3.3.1 Effects of pre- and post-weaning lard-rich diets on body weight, food and caloric intake, plasma glucose and FF A concentrations of male and female offspring ........................................................ 65

3.3.2 Effects of pre- and post-weaning lard-rich diets on plasma lipid levels of male and female offspring ........................... 68

3.3.3 Effects of pre- and post-weaning lard-rich diets on hepatic LDL-r mRNA expression in the male and female offspring ............ 73

3.3.4 Effects of pre- and post-weaning lard-rich diets on contractile responses of the male and female offspring aortas ...................................... 76

3.3.5 Effects of pre- and post-weaning lard-rich diets on endothelium -dependent

and -independent relaxation responses of the male and female offspring aortas ... 78

3.3.6 Effect of L-NAME on contractile responses of aortas in the male and female offspring .............................................. 81

3.4 DISCUSSION .................................. 83

3.5 LIMITATION OF THE STUDY ............ 90

CHAPTER FOUR ... 93

Effects of feeding a high-fat maternal diet rich in safflower oil (n-6 PUF A) during gestation and lactation on lipid metabolism and aortic vascular function in the adult offspring ... 93

4.1 INTRODUCTION .................................. 94

4.2 METHODS .............................................. 95

4.2.1 Experimental Design .................... 95

4.2.2 Quantitative-PeR analysis of hepatic LCAT and SR-B I mRNA expression. 97 4.2.3 Calculations and Statistical Analysis ................ 98

4.3 RESULTS ............................................ I 00 4.3.1 Effects of pre-and post-weaning safflower oil-rich diets on offspring body weight, food and caloric intake, and plasma concentrations of glucose and FF A .. 100

4.3.2 Effects of pre-and post-weaning safflower oil-rich diets on offspring plasma lipid levels ............................................ I 02 4.3.3 Effects of pre-and post-weaning safflower oil-rich diets on offspring hepatic mRNA expression ofLCAT and SR-81 ......................... 105

4.3.4 Effects of pre-and post-weaning safflower oil-rich diets on the contractile responses of the offspring aortas ............................ I 05 4.3.5 Effect of pre-and post-weaning safflower oil-rich diets on the endothelium- dependent and- independent relaxation responses of the offspring aortas ... 109

4.4 DISCUSSION .................. 112

CHAPTER FIVE ... 121

Comparison of a high-fat maternal diet rich in lard vs. safflower oil fed during gestation and lactation on lipid metabolism and aorlic vascular function in the adult offspring ... 121

5.1 INTRODUCTION ...................... 122

5.2 METHODS ................................. 123

5.2.1 Experimental Design ......................... 123

5.2.2 Calculations and Statistical Analysis ............... 123

5.3 RESULTS ............... 125

5.3.1 Effects of pre- and post-weaning high-fat diets enriched with lard or safflower oil on offspring body weight, food and caloric intake, and plasma glucose concentrations ..................... 125

5.3.2 Effects of pre- and post-weaning high-fat diets enriched with lard vs. safflower oil on offspring plasma lipid levels ................. 127

5.3.3 Effects of pre- and post-weaning high-fat diets enriched with lard vs. safflower oil on the contractile and relaxation responses ofthe offspring aortas ...... 130

5.4 DISCUSSION ................................ 134

CHAPTER SIX ... 140

Effects of pre- and post-weaning diets rich in different fatty acids on tissue fatty acid composition in the adult offspring ... 140

6.1 INTRODUCTION ................................... 141

6.2 METHODS .............................. 143

6.2.1 GLC analysis ............................ 143

6.2.2 Statistical analysis .......................... 143

6.3.1 Effects of pre- and post-weaning high-fat diets rich in lard vs. chow on

offspring hepatic fatty acid composition ................... 144

6.3.2 Effects of pre-and post-weaning high-fat diets rich in safflower oil vs. chow on offspring hepatic fatty acid composition .................................. 144

6.3.3 Effects of pre-and post-weaning high-fat diets rich in lard vs. safflower oil on the offspring hepatic fatty acid composition ............. 147

6.3.4 Effects of pre-and post-weaning high-fat diets rich in lard vs. chow on offspring heart fatty acid composition ...... 148

6.3.5 Effects of pre- and post-weaning high-fat diets rich in safflower oil vs. chow on offspring heart fatty acid composition ........ 151

6.3.6 Effects of pre- and post-weaning high-fat diets rich in lard vs. safflower oil on offspring heart fatty acid composition ...................... 151

6.4 DlSCUSSION ...................................... 155

CHAPTER SEVEN ... 163

Comparison of high-fat diets rich in flax oil vs. lard on the outcome of parameters associated with metabolic syndrome in adult SHRINDmcr-cp rat, a genetic model of metabolic syndrome ... 163

7.1 INTRODUCTION ........................... 164

7.2 METHODS ..................................................... 166

7.2.1 Ani1nals and diets ................................................ 166

7.2.2 Serum glucose and FFA analysis ............................ 167

7.2.3 Serum and hepatic lipid analysis .................. 169

7.2.4 Quantitative-PeR analysis ............................... 169

7.2.5 Oxidative stress analysis ............................... 171 7 .2.6 Statistical analysis .................................................... 171 7.3 RESULTS ............................................... I71

7.3.1 Effects of high-fat diet rich in flax oil vs. lard on body weight, organ weights, food and caloric intake in obese and lean SHRINDmcr-cp rats ......... 171 7.3.2 Effects of high-fat diets rich in flax oil vs. lard on TG and cholesterol

concentrations in serum and its lipoprotein fractions of obese and lean SHRINDmcr- cprats .................................................... 175 7.3.3 Effects of high-fat diets rich in flax oil vs. lard on hepatic TG and cholesterol concentrations in obese and lean SHRINDmcr-cp rats ......... 175 7.3.4 Effects of high-fat diet rich in flax oil vs. lard on hepatic gene expression in obese and lean SHRINDmcr-cp rats ............................. 179 7.3.5 Effects of high-fat diets rich in flax oil vs. lard on urinary TBARS levels in obese and lean SHRINDmcr-cp rats .............. 180 7.4. DISCUSSION ..................................... 184

CHAPTER EIGHT ... 190

Sztmmary and Conclusions ... l90

8.1 SUMMARY ............................. 191 8.1.1 Key Observations ......................... 192 8.2 IMPUCA TIONS AND FUTURE DIRECTIONS .................. 197

LIST OF TABLES

Table 1.1 Diagnostic criteria for metabolic syndrome according to the WHO and ATP III ......................... 4

Table 1.2 Diagnostic criteria of dyslipidemia by ATPIII ... 19 Table 2.1 Composition of the semi-synthetic diets used for a high-fat level. ...... 48 Table 2.2 Fatty acid composition of the experimental diets* .................. 49 Table 2.3 Pregnancy rate, pup survival rate and sex ratio of the offspring at the time of

weaning ... 52 Table 3.1 Body weight, food intake, caloric intake, plasma glucose and FF A concentrations of male and female offspring ... 66 Table 3.2 Half-maximal dose concentrations (pEC₅₀₎ of the male and female offspring aortas towards various drugs ..... 80 Table. 4.1 Sequence of the primers used for the quantitative PCR analyses ......... 99 Table 4.2 Body weight, food and caloric intake, plasma glucose and FF A concentrations of various offspring at the time of sacrifice ......... 10 I Table 4.3 Half-maximal dose concentrations (pEC50) of the offspring aortas towards various drugs ... 11 1 Table 5.1 Offspring body weight, food intake, caloric intake and plasma glucose concentrations ......... 126 Table 5.2 Half-maximal dose concentration (pEC5o) of the offspring aortas towards various drugs ....................................... 133 Table 6.1 Hepatic fatty acid composition of the offspring exposed to a high-fat diet rich in lard vs. chow during pre- and post-weaning time periods* ... 145 Table 6.2. Hepatic fatty acid composition of the offspring exposed to a high-fat diet rich in safflower oil vs. chow during pre-and post-weaning time periods* ... 146

Table 6.3. Hepatic fatty acid composition of the offspring exposed to a high-fat diet rich in lard vs. safflower oil during pre- and post-weaning time periods* ... 149 Table 6.4. Heart fatty acid composition of the offspring exposed to a high-fat diet rich in lard vs. chow during pre-and post-weaning time periods* ........... 150 Table 6.5. Heart fatty acid composition of the offspring exposed to a high-fat diet rich in safflower oil vs. chow during pre- and post-weaning time periods* ......... 153 Table 6.6. Heart fatty acid composition of the offspring exposed to a high-fat diet rich in lard vs. safflower oil during pre-and post-weaning time periods* ... 154 Table. 7. I Fatty acid composition of the experimental diets* ... 168 Table 7.2 Sequence of the primers used for the quantitative PCR analysis ....... 170 Table 7.3 Body weight, organ weights, food and caloric intake in obese and Jean SHR/NDmcr-cp rats fed high-fat diets rich in flax-oil vs. lard ....... 173 Table 7.4 Fasting serum glucose, FFA and insulin concentrations in obese and lean SHR/NDmcr-cp rats fed high-fat diets rich in flax oil vs. lard ... 174

LIST OF FIGURES

Fig. 1.1. Control of food intake under conditions of (A) low and (B) high levels of leptin

········································ 27 Fig.1.2 Lipid and lipoprotein metabolism ......... 35 Fig. 3.1 Experimental Design ... 63 Fig. 3.2 Plasma concentrations of (A) triglycerides and (B) total-cholesterol in the male and female offspring .......... 69 Fig. 3.3 Plasma concentrations of (A) LDL-cholesterol and (B) HDL-cholesterol in the male and female offspring ............. 71 Fig. 3.4 Plasma concentrations of (A) non-HDL cholesterol and (B) LDL/HDL- cholesterol ratio in the male and female offspring ...................... 72 Fig. 3.5 Real-time PCR analysis of hepatic LDL-r mRNA levels in the male and female offspring ................................. 74 Fig. 3.7 Dose-contraction responses of aortas from the male and female offspring to (A) KCI, ^(B) phenylephrine and (C) thromboxane mimetic U46619 ...... 77 Fig. 3.8 Dose-relaxation responses of aortas pre-constricted with U466l9, from the male and female offspring to (A) acetylcholine and (B) sodium nitroprusside ... 79 Fig. 4.1 Experimental Design ........ 96 Fig. 4.2 Plasma analysis of various offspring for (A) triglycerides, (B) total-cholesterol, (C) LDL-chole terol and (D) HDL-cholesterol. ........... I 03 Fig. 4.3 Plasma analysis of various offspring for (A) non-HDL chole terol and (B) LDLIHDL-cholesterol ratio ......... I 04 Fig. 4.4 Real-time PCR analysis of offspring hepatic ^(A) SR-B I and ^(B) LCAT mRNA expression ............ I 06 Fig. 4.5 Dose-contraction responses of the offspring aortas to (A) KCI, (B) phenylephrine and (C) thromboxane mimetic U46619 ....... 107

Fig. 4.6 Dose-relaxation response of aorta pre-constricted with U466l9 from various offspring, to (A) acetylcholine and (B) sodium nitroprusside ... II 0 Fig. 5.1 Experimental Design ............... 124 Fig. 5.2 Plasma concentrations of (A) triglycerides, (B) total-cholesterol, (C) LDL-

cholesterol and (D) HDL-cholesterol in various offspring ............... 128 Fig. 5.3 Plasma concentrations of (A) non-HDL cholesterol and (B) LDLIHDL-

cholesterol ratio in various offspring .............. 129 Fig. 5.4 Dose contraction responses of the offspring aortas to (A) KCI, (B) phenylephrine and (C) U46619 ..................... 131 Fig. 5.5 Dose-relaxation responses of the offspring aortas pre-constricted with U46619, to

(A) acetylcholine and (B) sodium nitroprusside ...... 132 Fig 7.1 TG concentrations in (A) whole serum (B), VLDL (C), LDL and (D) HDL fractions of obese and lean SHRINDmcr-cp rats fed high-fat diets rich in flax oil vs.

lard diet for 4-weeks ...................... 176 Fig. 7.2 Cholesterol concentrations in (A) whole serum (B), VLDL (C), LDL and (D) HDL fractions of obese and lean SHR/NDmcr-cp rats fed high-fat diets rich in flax oil vs. lard diet for 4-weeks ................. 177 Fig. 7.3 Hepatic (A) TG and (B) cholesterol concentrations in obese and lean SHRINDmcr-cp rats fed high-fat diets rich in flax oil vs. lard diet for 4-weeks .... 178 Fig. 7.4 Hepatic mRNA expression of (A) PPAR-a (B) PPAR-y and (C) SREBP-Ic in obese and lean SHRINDmcr-cp rats fed high-fat diets rich in flax oil vs. lard diet for 4-weeks .................................................. 181 Fig. 7.5 Correlation analysis of hepatic PPAR-y mRNA expression with (A) hepatic TG

and (B) cholesterol concentration in obese and lean SHRINDmcr-cp rats fed high-fat diets rich in flax oil vs. lard diet for 4-weeks ................ 182

AA ABCA-1 ALA ATP-III Apo ACh ANOVA BMI CETP CYP-71 CVD DBP DOH aD DHA EPA EDHF

ER

EDTA FFA GTT GAPDH HDL GLC IDL KCL LA LDL

LIST OF ABBREVIATIONS

Arachidonic acid

A TP-binding cassette transporter protein-) a-linolenic acid

Adult Treatment Panel III Apolipoprotein

Aetylcholine

Analysis ofvariance Body mass index

Cholesteryl ester transfer protein Cholesterol 7 alpha-hydroxylase Cardiovascular disease

Diastolic blood pressure

Developmental origins ofhealth and disease Docosahexaenoic acid

Eicosapentaenoic acid

Endothelial derived hyperpolarizing factor Endoplasmic reticulum

Ethylene diamine tetra acetic acid Free fatty acids

Glucose tolerance test

Glyceraldehyde 3-phosphate dehydrogenase High-density lipoprotein

Gas -liquid chromatography Intermediate-density lipoproteins Potassium chloride

Linoleic acid

Low-density lipoprotein

LCAT LDL-r LPL L-NAME MUFA NAFLD NO NOS PE PSS PPAR PUFA PLTP PI-3 Kinase PAR

RXR

RNA ROS SBP SHR SFA SR-BI SNP SE SD SREBP TZD TG TX-A2

Lecithin: cholesterol acyltransferase Low-density lipoprotein receptor Lipoprotein lipase

N (G)-nitro-L- arginine methyl ester Mono-unsaturated fatty acids

Non-alcoholic fatty liver disease Nitric oxide

Nitric Oxide synthase Phenylephrine

Physiological salt solution

Peroxisome proliferators-activated receptor's PPAR Polyunsaturated fatty acids

Phospholipids transfer protein Phosphoinositide-3 kinase Predictive adaptive response Retinoid X receptor

Ribonucleic acid

Reactive oxygen species Systolic blood pressure

Spontaneously hypertensive rats Saturated fatty acids

Scavenger -receptor B I Sodium nitroprusside Standard error of mean Standard deviation

Sterol-regulatory element binding protein Thiazolidinedione

Triglycerides Thromoboxane A₂

Type 2OM VSMC VCAM-1 VLDL WHO

Type 2 diabetes mellitus Vascular smooth muscle cells Vascular cell adhesion molecule-]

Very-low density lipoprotein World health organization

LIST OF APPENDICES

APPENDIX I ..................................... 258 APPENDIX II ................................. 259

r - -- - - - - - -

CHAPTER ONE

Introduction and Overview

1.1 CARDIOVASCULAR DISEASE

Cardiovascular disease (CVD) is a complex and multifaceted disease that currently represents the major cause of deaths worldwide. The World Health Organization (WHO) attributed 30% of all global deaths (i.e. 15.3 million) as well as 10.3% of the total disability adjusted life years (DALY s) lost in 1998 to CVD (WHO, 1999). The Heart and Stroke Foundation of Canada reported a similar mortality rate in 2005, where 31% of deaths occurred due to CVD (Statistics Canada, 2009). The global burden of CVD affects all sections of society. Cardiovascular deaths in 1998 contributed to 34% of global mortality in women and 28.2% of all deaths in men (WHO, 1999). Once considered to be the 'disease of affluence' CVD is now shown to affect both developed and developing nations (Reddy and Yusuf, 1998). According to the Global Burden of Disease Study, it has been projected that a 55% rise would occur in DALY loss attributable to CVD between 1990 and 2020 in developing countries (Murray and Lopez, 1996). In addition to the increasing incidence of CVD, the early age at which it manifests would also contribute to the burden of CVD. It is projected that 6.4 million deaths would occur due to CVD in the age group of 30-69 years in developing countries by 2020 (Murray and Lopez, 1996). This rapid rate of change, together with the increasing burden of disease, is creating a major public health threat, which demands immediate and effective action.

Besides the social burden, there are data available to indicate the economic burden of CVD. According to the American Heart Association and the American Stroke Association (2006), total direct and indirect costs of CVD and stroke were estimated to

be $403.1 billion for the year 2006. At the same time, European Union reported an annual cost of €169 billion (Leal eta!., 2006), while the Heart and Stroke Foundation of Canada reported an annual expenditure of $22.2 billion to be associated with the incidence of CVD (Canadian Heart Health Strategy-Action Plan Steering Committee, 2009). The overall social and economic burden of CVD points toward the need for action to strengthen the preventive measures to counter the spread of CVD epidemic, which is now being widely recognized by many countries. The World Health Assembly adopted a resolution that urged Member States to collaborate with WHO to develop " ... a global strategy on diet, physical activity and health for the prevention and control of non- communicable diseases, based on evidence and best practices, with special emphasis on an integrated approach ... " (WHO, 2002).

A number of chronic conditions such as dyslipidemia, visceral obesity, hypertension, glucose intolerance and type 2 Diabetes Mellitus (DM) are recognized as independent risk factors for the development of CVD. The co-existence of three or more of these conditions can also be identified as 'metabolic syndrome', which can double the risk of subsequent development of CVD and premature death (Bricker and Greydanus, 2008). The diagnostic criteria for metabolic syndrome by WHO (Alberti and Zimmet, 1998) and National Cholesterol Education Program- Adult Treatment Panel III (NCEP- ATPIII) (NCEP- Expert Panel 2001) (Grundy eta!., 2005) is given in Table 1.1.

Diet and nutrition have long been identified to be the key players m the development of CVD and its risk factors. It is apparent at the global level that 'nutrition

Table 1.1 Diagnostic criteria for metabolic syndrome according to the WHO and ATP III

Component

Abdominal/central obesity Hypertriglyceridemia

WHO crtieria Onsulin resistance*

plus two of the rest)

A TP ni criteria (Three of the following)

Waist to hip ratio: > 0.90 (men),> 0.85 Waist circumference:> I 02 em in (women), or BMJ > 30 kglm² men,> 88 em in women

>= 150 mgldL (>= 1.7 mM) >= 150 mgldL

Low HDL cholesterol < 35 mgldL (< 0.9 mM) for men,< 39 < 40 mgldL (<1.036 mM) for men, mgldL ( <1.0 mM) for women <50 mgldL ( <1.295 mM) for women - High blood pressure >= 140/90 mm Hg or documented use >= 130/85 mm Hg or documented

High fasting glucose

Microalbuminuria

of antihypertensive therapy use of antihypertensive therapy impaired glucose tolerance, impaired

fasting glucose, insulin resistance, or diabetes

Urinary albumin to creatinine ratio: 30 mglg, or albumin excretion rate: 20

meg/min

>= II 0 mgldL (>= 6.1 mM)

(Alberti and Zimmet, 1998; Grundy et al., 2005)

WHO = World Health Organization; ATP = Adult Treatment Panel; BMI = body mass index; HDL =high-density lipoprotein.

*Insulin resistance is identified by type 2 DM or impaired fasting glucose.

transition' has swept the entire world since the second half of the twentieth century inducing major modifications in diet, which has paralleled the rise in the incidence of CVD. The dietary changes that characterize the 'nutrition transition' include both quantitative and qualitative changes in the diet. The adverse dietary changes include shifts in the structure of the diet towards a higher energy-density diet with a greater role for fat and added sugars in foods, greater saturated fat (SF A) intake, reduced intakes of complex carbohydrates and dietary fiber, and reduced fruit and vegetable intakes (Drewnowski and Popkin, 1997). These dietary changes are compounded by lifestyle changes that reflect reduced physical activity at work and during leisure time.

While it is apparent that nutrition during adult life is important, there is increasing evidence that CVD risks begin in fetal life and continue into old age (Barker, 1995;

1997a; 1997b, 2004a; 2004b ). Adult CVD has therefore been identified to reflect cumulative differential lifetime exposures to damaging physical and social environments.

For these reasons, the Joint WHO/Food and Agriculture Organization Expert Consultation committee adopted a life-course approach to capture the cumulative risk as well as many opportunities for dietary intervention. While accepting the imperceptible progression from one life stage to the next, five stages were identified for converuence, which were recognized as 1 ). Fetal development and the maternal environment, 2) Infancy 3), Childhood and adolescence 4). Adulthood, and 5), Aging and older people.

Nutritional and lifestyle interventions at each stage of this life-course approach were recognized as an important strategy to control the global epidemic of CVD by the Expert Consultation Committee (WHO, 2003).